Adhesins of Enterotoxigenic <i>Escherichia coli</i> Strains That Infect Animals

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Adhesins of Enterotoxigenic Escherichia coli Strains That Infect Animals

doi: 10.1128/ecosal.8.3.2.1.2

DIETER M. SCHIFFERLI

[SECTION EDITOR: JAMES P. NATARO]

Posted March 29, 2005

University of Pennsylvania Veterinary School, 3800 Spruce Street, Philadelphia, PA 19104-6049

Phone: 215-898-1695 ; Fax: 215-898-7887; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

INTRODUCTION

The first described adhesive antigen of Escherichia coli strains isolated from animals was the K88 antigen (212), expressed by strains from diarrheic pigs. K88, which originally was thought to be a capsular antigen ("K" for "Kapsel," the German word for capsule), was later shown to be a protein antigen whose expression depended on the presence of a plasmid (264). The K88 antigen was visible by electron microscopy as a surface-exposed filament that was thin and flexible and had hemagglutinating properties (263). Like other nonflagellar hairlike appendages on the bacterial surface, K88 fimbriae were also called pili; however, the designation of fimbriae is preferred by some because of historical precedence (70) or to distinguish adhesive (fimbriae) from conjugative (pili) organelles. That bacteria (147), and particularly E. coli (105), can agglutinate erythrocytes and that this property can be mediated by fimbriae (71), have been described previously. However, the fact that intestinal adhesion and colonization of E. coli in diarrheic animals (254) is mediated by fimbriae was first shown with K88 fimbriae (252). More importantly, the role of fimbriae as virulence factors was first demonstrated with K88 fimbriae (253). In a seminal paper, Smith and Linggood infected pigs with an enterotoxigenic E. coli (ETEC) strain that had either its enterotoxin- or its K88 antigen-determining plasmid missing to demonstrate that both the enterotoxin and K88 were needed to elicit severe diarrhea or death by dehydration. Moreover, in the absence of K88, significantly fewer E. coli could be isolated from the intestines, the difference being most impressive in the proximal portions of the small intestine. In this extensive investigation, the authors applied all the basic approaches for the study of bacterial virulence factors that were later reformulated as the molecular Koch's postulates (83). For example, intestinal colonization and the induction of diarrhea in neonatal or weaned pigs were studied with a variety of strains, including plasmid-cured porcine ETEC strains into which the same or similar plasmids were reintroduced. Alternatively, nonpathogenic isolates were rendered pathogenic by introduction of the corresponding plasmids. To support their results, the authors even undertook competition experiments between the same strain carrying or missing either one or both plasmids. Additional studies by Jones and Rutter confirmed the role of K88 in ETEC adhesion and colonization of the small intestines of piglets (138). The latter study yielded data on the antiadhesive properties of K88-specific antibodies. This result led the authors to an ingenious experiment in which they demonstrated that neonatal piglets could be protected against ETEC by passive immunity acquired by colostrum from an immunized mother sow (233).

Finally, the K88 gene cluster was the first fimbrial system shown to be expressed and functional when cloned into a K-12 strain of E. coli (175). This work and further studies on both the K88ac (140) and K88ab (176) genes and their products spurred the cloning (114) and genetic study of other fimbriae, such as the common type 1 and the P fimbriae (205, 210).

FIMBRIAE INVOLVED IN ETEC PATHOGENESIS

By definition, ETEC produce two types of virulence factors responsible for diarrhea: fimbrial adhesins and enterotoxins. Many different fimbriae have been identified in animal ETEC, as shown in Table 1. The role of these fimbriae in the pathogenesis of ETEC has been best studied with K88, K99, 987P, and F41. However, even with these fimbriae, the experiments that supported their essential roles in the pathogenesis of ETEC all used clinical strains cured of plasmids encoding the fimbrial genes. Confirming experiments with defined isogenic mutants were never undertaken (83). Thus, it can be argued that the absence of other plasmid-encoded genes could have affected the results. Nevertheless, extensive additional evidence supports the importance of the K88, K99, 987P, and F41 fimbriae as essential adhesive virulence factors of animal ETEC (42, 126, 127, 128, 180, 181, 183, 184, 186, 188, 189, 195, 198, 199, 232, 276, 277). Evidence supporting the importance of the other fimbriae in Table 1 as adhesive virulence factors of ETEC is mainly epidemiological and indirect (e.g., passive protection with anti-F18 or F17 antibodies) (51, 116, 305), since nonfimbriated plasmid-less or isogenic mutants have yet to be studied in animals. All the fimbriae in Table 1 have been found associated with enterotoxin (genes) on clinical isolates of animals with diarrhea. However, F17 fimbriae (including F17b, F17c, and F17d) are also expressed by diarrheagenic non-ETEC strains or by extra-intestinal E. coli (21, 66, 150). Clinical isolates of ETEC have clonal properties, since there is a preferential association of fimbriae with certain enterotoxins and O-serotypes (Table 1) that has lead to their classification into major pathotypes (107). The same isolate frequently carries the genes to express two or more fimbriae. Each fimbrial type carries at least one adhesive moiety that is specific for a certain host receptor, determining host species, age, and tissue specificities (Table 1).

Table 1Fimbriae of animal enterotoxigenic E. coli

Some fimbriae are not included in Table 1 because their involvement in the pathogenesis of ETEC remains unknown. The CS1541 fimbriae, for example, are expressed in vivo by porcine ETEC strains (33, 34), but they do not bind to porcine enterocytes in vitro. Other fimbriae such as the F165(1) (similar to Prs or F11), F165(2) (similar to F1C), and CS31A (protein capsule) fimbriae have been reported to be associated with animal ETEC strains (producing ST). These fimbriae, however, are mainly expressed on non-ETEC strains of porcine or bovine origin (20, 50, 81, 302). Most isolates expressing these fimbriae do not coexpress enterotoxins but more typically express a cytotoxic necrotizing factor (CNF) or, in some cases, the enteroaggregative E. coli heat-stable toxin (EAST-1, a diarrheagenic toxin). The CS31A fimbriae do not mediate bacterial adhesion to bovine or porcine enterocytes or their brush borders (94). Whether non-ETEC binding to enterocytes can be mediated by CS31A variants is not clear (166). No enteroadhesive property in relation to farm animals has been reported for both F165 fimbriae. Thus, both F165 and CS31A lack the typical properties of ETEC fimbriae. Some Dr-like fimbriae (Afa-7 and Afa-8) are expressed by diarrheagenic E. coli that are non-ETEC or extra-intestinal E. coli (150, 160).

Epidemiology

According to the last three National Swine Surveys in the United States, diarrhea remains a major cause of mortality and morbidity (279, 280, 281). ETEC are the most frequently diagnosed pathogens among neonatal and postweaning piglets that die of diarrhea (80, 181). ETEC isolated from neonatal piglets express typically K88, K99, or 987P fimbriae (Table 2), whereas postweaning ETEC isolates express mainly F18 and/or K88 fimbriae (1, 53, 63, 182, 196, 215, 223, 300). The prevalence of the three major fimbriae expressed by ETEC strains that colonize the intestines of neonatal piglets shows both temporal and geographic variation (Table 2). Although K88 became the major fimbriae of newborn piglet ETEC in the United States, 987P-fimbriated ETEC remain the major problem in some other countries. In recent years, fimbriated isolates of sick pigs presented a less diverse K88- and O-serotype profile than 20 to 30 years ago. For example, the K88ac variant became even more predominant than the other K88 variants and O149 became a major O-serotype of ETEC in America and Europe (91, 96, 237, 296, 300). It has been suggested that the observed variations of these serotypes over time are the result of vaccination pressures. Alternatively, successful selection of certain serotypes might be due to the changes the pig-farming industry has undergone over the years (fewer and larger facilities). However, the adaptation of animal ETEC is not exclusively clonal (214).

Table 2Numbers of K88-, K99-, and 987P-fimbriated ETEC isolated from piglets in various studies since 1979

Fimbrial Genes

Like most studied bacterial fimbriae, the production of ETEC fimbriae requires sets of genes that are organized in clusters that include one or more operons (Fig. 1). However, unlike the well-studied type 1 or P fimbriae of E. coli, most ETEC fimbrial gene clusters are located on plasmids. These plasmids are quite large (40 to 100 kb) and usually also encode enterotoxin genes (19, 84, 88, 130, 158, 159, 240) (Table 1). In some cases, the fimbrial genes are adjacent to an enterotoxin gene, creating a pathogenicity islet that includes all the genetic determinants responsible for the symptoms of diarrhea (145, 241). However, ETEC strains typically carry several large plasmids encoding additional enterotoxin genes and fimbrial systems, suggesting that the resulting redundancy of colonization and diarrheagenic factors must be beneficial to the survival or the transmission of ETEC.

Fig. 1Genetic organization of animal ETEC fimbrial gene clusters. Genes encoding similar products or products with similar functions were labeled with the same pattern; genes for the major fimbrial subunit (black), minor fimbrial subunits (diagonal lines), minor adhesive subunit (gray), chaperones (horizontal lines), usher (vertical lines), regulators (checkered), and mobile or conjugation elements (white).

As documented with the type 1 and P fimbriae, the fimbrial gene clusters of animal ETEC (Fig. 1) also encode proteins that have one of three essential functions for the production of fimbriae (170). First, two or more genes encode the structural components of the fimbriae. One protein, the major fimbrial subunit, forms most of the polymeric structure of the fimbriae, whereas the other components are incorporated as minor subunits. One (or sometimes more) subunit(s) of each fimbria carries at least one binding site for a specific mammalian host receptor. Second, the fimbrial biogenesis machineries all consist of two types of molecules, one or more periplasmic chaperones and one outer membrane protein or usher (271). Finally, the best-studied K88, K99, and 987P gene clusters encode regulatory proteins that are fimbria specific. A model depicting the subcellular location of the 987P subunits, chaperones, usher, and regulatory protein required for fimbrial biogenesis is shown in Fig. 2.

Fig. 2A fimbrial biogenesis model. The 987P fimbriae consists of the major subunit FasA and two minor subunits, the adhesin FasG and the linker subunit FasF. The usher protein FasD locates in the outer membrane and serves as a subunit channel. 987P expression involves three periplasmic chaperones: the FasA-specific chaperone FasB, the FasG-specific chaperone FasC, and FasE, a chaperone-like protein that facilitates FasG export. Whether FasF interacts with any of these chaperones is not known. FasH (FapR) is an AraC-like transcriptional activator of fasA.

Fimbrial Structure

The hairlike appearance of fimbriae is best observed by negative staining electron microscopy. Fimbriae, which can reach 2 μm in length, have been typically classified by their thickness (diameter), which varies from 3 to 7 nm. K88, F41, and F17 are reported to be the thinnest, and 987P is the thickest ETEC fimbria, whereas K99 and F18 are intermediate in diameter. Diameter values cited in the literature may vary according to the bacterial growth conditions and the staining techniques used by each investigator (69, 106, 170, 245). One fimbrial thread or fimbria consists of the spiral arrangement of hundreds of protein subunits along a filamentous axis (32). The less the subunits are compacted along the axis, the less they share surfaces of interactions and the thinner and more flexible the fimbriae appear. A helpful model for thick or thin fimbriae is a helix that is compressed or stretched apart, every helix turn touching the next turn only in the compressed structure, and the bending of the whole filamentous axis encountering the least amount of resistance in the stretched form. The model is also consistent with an axial hole (~2 nm), which is only visible on the electron micrograph of a thicker fimbria. Thick fimbriae can be stretched under certain in vitro conditions (3, 36). It has been suggested that fimbrial stretching occurs in vivo to adjust and coordinate the lengths of the few hundred fimbrial threads anchoring the colonizing bacteria that are submitted to the intestinal peristalsis and its resulting shear force (255). Fimbria-mediated bacterial adhesion to a target cell is enhanced by shear force, as described with the F41 fimbriae (35). An elegant study with the type 1 fimbriae showed that this enhancement involved the extension of the interdomain linker chain in the adhesive fimbrial subunit (275). Whether shear stress induces other conformational changes in the fimbriae remains to be determined. The components of animal ETEC fimbriae are listed in Table 3. Immune electron microscopy of animal ETEC fimbriae usually shows that the minor subunits are located at the fimbrial tips and at discrete sites along the fimbrial threads (40). Whether minor subunits are effectively incorporated into a fimbrial body or easily break off from fimbrial tips and then stick along the sides of the fimbrial threads remains controversial. Mechanical fragmentation of isolated type 1 fimbriae of E. coli resulted in increased adhesin-mediated binding, suggesting the uncovering of hidden or incorporated adhesive minor subunits (222).

Table 3The structural proteins of animal enterotoxigenic E. coli

Fimbrial Adhesins and Host Receptors

In general, it is assumed that the fimbriae of ETEC strains act only as anchoring devices to serve bacterial colonization of the intestinal surface. This assertion is supported by the absence of reported visible cytoskeletal rearrangements of intestinal epithelial cells to which ETEC have bound. The possibility of fimbria-mediated signal transduction mechanisms leading to other cellular responses, as shown with non-ETEC pathogens (90, 101), however, has not been studied extensively with enteroadhesive ETEC strains and small intestinal epithelial cells. Unknown is the potential of ETEC strains to optimize the delivery of flagellin (248) or other bacterial effector molecules to the mucosal innate immune system of the small intestine. The pathogenesis of ETEC strains is specifically linked with the colonization of the small intestine and not of the large intestine. The distribution of the receptor(s) and the differential environmental signals regulating fimbrial expression in each intestinal segment (74, 76) determine the bacterial colonization sites.

Many fimbriae mediate hemagglutination. Since fimbriae most frequently act like lectins by binding to the carbohydrate moieties of glycoproteins or glycolipids, fimbrial receptors have frequently been studied with red blood cells of various animal species. Although hemagglutination remains a convenient way to study and classify fimbriae (25, 37, 103), some fimbriae of animal ETEC such as 987P do not agglutinate red blood cells or do so only after chemical treatment (115). Another caveat is that the O- and N-glycosylation profiles and the glycosylated host molecules on red blood cells might be quite different from the ones found in the intestinal mucus and on enterocyte brush borders of the relevant animal species. The presence, modification, or absence of some of these receptors in the mucus or on the brush borders varies with age, and these changes have been proposed to explain age-dependent intestinal colonization and ETEC-mediated diarrhea. For example, only newborn and weaned piglets had K88 receptors in their mucus, the latter more than the former (52), while these receptors were hardly detectable in the mucus of 6-month-old pigs (298). Similarly, intestinal cells from older pigs or calves were resistant to K99-mediated adhesion (231). This correlated with the age-dependent disappearance of the N-glycolyl group in intestinal glycolipids required for the K99 receptor activity (269, 304). Studies on the 987P receptors suggested that intestinal sulfatide (the major 987P brush-border receptor) is released in the mucus of postneonatal pigs inhibiting fimbria-mediated adhesion and colonization (58, 62, 65). In contrast, the adhesion and colonization by F18-fimbriated ETEC isolates was suggested to be dependent on receptors that develop progressively with age during the first 3 weeks after birth (197). F17-mediated bacterial binding to ileal mucus of older calves was decreased when compared with the binding to mucus of younger animals (191). However, the age-specific presentation of intestinal receptor molecules alone does not determine the susceptibility of neonatal or weaned animals to fimbria-mediated colonization. Both receptor expression, determined by the genetic makeup of an individual animal, and the protection provided by colostral antibodies also play a role. The major intestinal receptors for animal ETEC fimbriae and their cognate fimbrial adhesins are listed in Table 4.

Table 4The fimbrial adhesins of enterotoxigenic E. coli and their receptors

Many different receptors have been described for the K88 fimbriae and its serological variants (Table 4). That the antigenic classification of the K88 variants also determines their binding particularities (16, 266) is consistent with the identification of the major fimbrial subunit FaeG as the K88 adhesin (132, 135). Substituting the phenylalanine at position 150 of FaeG for a serine drastically reduced the hemagglutinating property of the K88ab fimbriae, suggesting that this residue might also be important for intestinal binding (132). The K88 receptor list in Table 4 is not exhaustive, other receptors of various molecular weights having been reported for the K88 fimbriae, as discussed in several reviews (27, 136, 286). Some of the additional mucin receptors might represent released degradation products of larger brush-border receptors. Depending on the presence or absence of the different K88 receptors, six pig phenotypes (labeled A to F) have been described (Table 5). More recently, the existence of a C phenotype has been questioned (27). The genetic loci for the bc and b receptors of the K88 fimbriae were located on porcine chromosome 13 (224). As shown with different glycoconjugate receptors, the three K88 variants demonstrate lectin activities specific for a minimal recognition sequence containing a β-linked HexNAc, a terminal β-linked galactose enhancing the binding (99). The context of this sequence on the different receptors most likely is responsible for the binding specificities of the K88 variants. All the intestinal ceramides that act as receptor for the K88, K99, and 987P fimbriae need to be hydroxylated (142, 221, 304), indicating the importance of the lipid moiety in the binding properties of gangliosides with short carbohydrate chains. Moreover, the membrane-embedded lipid portion of a glycolipid receptor determines the orientation of the carbohydrate target on the surface of host cells and thus plays an essential role in the recognition by a fimbrial lectin (139). DNA hybridization and gene expression studies indicated that the F41 fimbrial gene cluster is most similar to the K88 gene cluster, with the exception of the major subunit gene (6, 190). Thus, it is assumed that this fimbrial subunit acts as the F41 adhesin. An intestinal receptor for the F41 fimbriae remains to be identified, although it might include N-acetylglucosamine in a carbohydrate group that mimics one on glycophorin A^M, as determined by hemagglutination assays (35, 153).

Table 5Porcine phenotypes and their K88 receptors (12, 24, 27, 110, 295)

Similar to the K88 fimbriae, the major K99 subunit (FanC) was shown to be responsible for the hemagglutinating properties of the fimbriae (133, 134). Site-directed mutagenesis of two positively charged residues, lysine 132 and arginine 136, affected the interaction with erythrocytes known to share some of the sialylated glycolipid receptors with piglet and calf intestines (149, 209, 251, 269, 270, 304).

In contrast to K88 and K99, 987P fimbriae do not agglutinate mammalian red blood cells but only bind to intestinal cells (58, 59, 60, 61, 62). The 987P minor subunit FasG binds specifically to intestinal porcine proteins of 32 to 35 kDa that remain to be characterized (62, 143). FasG also mediates 987P binding to a glycolipid receptor, porcine intestinal sulfatide (65, 142). Out of twenty single arginine or lysine to alanine mutants, binding to sulfatide-containing liposomes was reduced in four cases (residues 17, 116, 118, and 200) and abrogated for one mutant (lysine 117). All five mutants produced wild-type levels of 987P fimbriae. It was proposed that one ore more of these residues communicate with the sulfate group of sulfatide by hydrogen bonding and/or salt bridges (48). All the allelic FasG proteins with reduced binding to sulfatide still interacted like wild-type FasG with the protein receptors of porcine brush borders. At least two segments of FasG that did not include lysine 117 were involved in this interaction, suggesting that different residues, and thus different domains of FasG, are required for binding to the protein and the sulfatide receptors (47). In addition to the two enteroadhesive properties of FasG, a third type of 987P binding occurs between the major subunit FasA and piglet brush-border hydroxylated ceramide monohexoside (142).

For the F18 fimbriae, the minor subunit FedF of F18ab (120) was shown to act as an adhesin specific for porcine intestinal epithelial cells (249). The continuous FedF sequence from residue 60 to 109 was shown to be important for binding (250). Each of its charged residues was substituted for alanine, one at a time, and three mutations (replacing lysine 72, histidine 88, or histidine 89) significantly diminished bacterial binding to jejunal epithelial cells. Binding was abolished with a double mutant (lysine 72 and histidine 88) and the triple mutant. All these mutants produced wild-type levels of fimbriae. The fedF sequence of 15 clinical isolates revealed 97% identity, with eight sequences showing an asparagine substitution to glycine or aspartic acid at position 73 (250). Both substituted and "wild-type" alleles were found in O139-serotyped strains, which typically express F18ab, and in O141-serotyped isolates, which usually express F18ac. Thus, it is likely that F18ab and F18ac bind to the same receptor(s), as suggested in earlier studies showing that either F18ab or F18ac fimbriae inhibit F18-mediated bacterial attachment to enterocytes (226). Whether the adhesive function of F18 fimbriae is modulated by the reported allelic sequence profiles of fedF in clinical isolates remains an open question. Genetic studies have shown that susceptibility to F18ab-mediated enteroadhesion was inherited in pigs as a dominant trait and that it was linked to the α(1,2)-fucosyltransferase FUT1 gene on porcine chromosome 6 (164, 165, 294). These studies suggested that this glycosylase adds an essential fucose to one or more glycoconjugate receptors for the F18ab fimbriae. The identity of this receptor(s) remains to be determined.

The F17-G minor fimbrial subunit is the adhesin of the F17 fimbriae (155). F17-G deletion mutants produce normal fimbriae that do not bind to calf intestinal epithelial cells. Binding inhibition assays suggested that the carbohydrate specificity of the F17a fimbriae is a terminal or an internal N-acetylglucosamine on O-linked oligosaccharides of bovine mucins or intestinal glycoproteins (191, 235). The crystal structures of the immunoglobulin-like lectin domains of two F17 adhesins (F17-G and the related adhesin GafD) that bind N-acetylglucosamine were resolved (38, 167).

It is curious that animal ETEC expression of multiple ligands on one fimbrial organelle has only been observed with 987P. The potential of 987P to adhere to intestinal cells by creating several receptor-ligand bridges is consistent with Karlsson's proposed model involving a succession of primary and secondary binding sequences of events acting synergistically for successful bacterial colonization (139). Alternatively, the presence of different subunits with various binding properties on the same structure could permit fimbriated ETEC to use an energetically economical system for temporally or anatomically separated ligand-receptor activities. Since the attachment of the K88 and K99 mutants were never studied with intestinal receptors, it remains possible that, similarly, one (or more) of their minor subunits (Table 3) mediates an enteroadhesive function that has not yet been discovered.

Fimbrial Biogenesis

To produce fimbriae on the surface of ETEC, the structural subunits have to be transported directionally through two membranes. With the exception of the fimbria-specific regulatory proteins, both the fimbrial subunit and biogenesis proteins have typical signal sequences allowing them to cross the cytoplasmic membrane by using the general secretory pathway (262). Having reached the periplasm, the exported fimbria-specific chaperone(s) associate with the fimbrial subunits that appear on the periplasmic side of the inner membrane (178). The subunits are then delivered to the usher protein on the periplasmic side of the outer membrane. The chaperones protect the fimbrial subunits against proteolytic degradation and premature assembly in the periplasm. Most importantly, chaperones keep the subunits in an export-competent conformation, the energy for subunit export and assembly being maintained by the conformational state of the chaperone-associated subunits (131). Subunit release and delivery to the usher by the chaperone is coupled with the assembly of the subunit into a fimbrial fiber (18). The usher molecule forms homopolymeric central channels that are required for the linear translocation of fimbrial subunits through the outer membrane (178, 272, 273), resulting in the incorporation of subunits into the growing fimbrial helix on the bacterial surface (238). Thus, the usher also acts as an anchor for the elongating fimbrial fiber (140). Minor fimbrial subunits are frequently observed on the tip of fimbriae by immune electron microscopy. Since fimbriae grow from the base (157), tip-associated minor subunits have to be delivered to the usher before the major subunits. The role of these minor subunits can be essential in initiating fimbrial elongation, as described below for the individual fimbriae, when a mutation in a minor subunit gene results in the reduction or lack of fimbriation.

DNA sequence differences of the K88ab, K88ac, and K88ad fimbriae are found in their major subunits FaeG. However, the sequences of the accessory genes of the K88ab and K88ac are identical (13, 170). The K88 chaperone, a homodimer of FaeE (14, 169, 171, 173), and the usher FaeD (108, 174, 282) are involved in the export of the adhesive major subunit FaeG and three to five minor subunits (FaeC, FaeF, FaeH, and possibly FaeI and FaeJ) (68, 178, 179, 286, 292). The FaeC subunit is located at the tip of the fimbria and plays an essential role in initiating fimbrial export and assembly, since there is no fimbriation in its absence (177, 219). FaeC, unlike FaeG, FaeH, and FaeF, interacts only weakly or indirectly with the chaperone FaeE in the periplasm but binds well to the usher FaeD (172). The FaeF and FaeH subunits locate at distinct distances along the fimbrial length and are involved in fimbrial biogenesis; 40 to 100 times less fimbriae are expressed in their absence (17). Genetic studies predict that the usher FaeD spans the outer membrane with 22 β-strands, leaving relatively long N- and C-terminal ends in the periplasm (108, 282). The FaeI and FaeJ proteins share sequence similarities with the other subunits. However, their roles are not clear and the corresponding mutants have no altered phenotypes (17). The F41 biogenesis apparatus has not been studied in great detail, but it has been shown that the F41 gene cluster contains a gene arrangement similar to the K88 gene cluster (6, 190). Moreover, F41 subunit-containing fimbriae were expressed by complementing its gene with the accessory genes from the K88 gene cluster, indicating that the F41 and K88 export and assembly systems are closely related (146).

The export of the K99 subunits is coordinated by the monomeric chaperone FanE (14, 168) and the usher FanD (56). The minor subunit FanF is positioned both at the tip and along the fimbrial shaft (246). This is consistent with the phenotype of a fanF mutant that produces only 0.1% of the wild-type number of fimbriae, all being shorter than wild type. FanG and FanH are also minor components of the K99 fimbriae (229, 247). They associate with FanF and also participate in the initiation and elongation of the fimbriae, since fanG and fanH mutants are nonfimbriated or produce shorter and fewer fimbriae (1 to 2% of wild-type levels).

FasG, an adhesive minor subunit, is the first exported subunit of the 987P fimbria, followed by FasF, the second minor subunit proposed to act as a linker molecule, and FasA, the major structural subunit (40, 143). FasG and FasF can be visualized as well at the tip as along the fimbrial shaft. In their absence, no fimbriae (or very rare short fibers for a fasF mutant) are expressed (241, 242). The outer membrane protein FasD is the 987P usher protein and mutagenesis studies suggest that its structure consists of a β-barrel with 28 amphipathic β-strands crossing the membrane (239). FasD was most accessible to proteases from the periplasmic side, implying the presence of a membrane-embedded usher with large periplasmic loops. In contrast to most other fimbrial systems, 987P fimbrial biogenesis involves three different chaperones (72). FasB is the chaperone associating with the major subunit FasA, whereas FasC acts independently of FasB as the FasG-specific chaperone. FasE, a chaperone-like protein was also located in the periplasm. Although no FasE-associated Fas protein could be detected, FasE was shown to be required for optimal export of FasG.

Major subunit alleles of the F18ac and F18ab fimbriae have been studied (30, 119, 141). Only the accessory genes and products of the F18ab fimbriae have been investigated. However, by analogy to the K88 fimbriae, it is assumed that the F18ab and F18ac accessory genes have the same or very similar sequences. Based on their sequences, the fedB and fedC genes were proposed to encode the usher and periplasmic chaperone proteins of the F18ab fimbriae (249). This fimbrial biogenesis apparatus is needed for the export and assembly of the three F18ab subunits, FedA, the major component (117), as well as the two minor fimbrial subunits FedE and the adhesin FedF (120) Although both FedE and FedF are nonessential for fimbriation, fedE and some fedF mutants formed longer fimbriae, indicating that their products inhibit fimbrial elongation. Although the FedF protein carries the adhesive moiety of F18ab, fedE mutants (like fedF mutants) do not bind to porcine intestinal villi, suggesting that FedE is either involved in the export and assembly of FedF or that it modulates the binding properties of FedF on F18.

Analysis of the DNA sequence from the F17a gene cluster of a bovine ETEC strain revealed the presence of four genes (GenBank accession number AF022140) (155). The chaperone F17a-D and the usher protein F17a-C are essential for the export and assembly of the major subunit F17a-A and the minor adhesive subunit F17a-G. An F17a-G mutant can make wild-type levels of unaltered fimbriae that do not bind, indicating that the F17a-G adhesin is not required for fimbriation. A major subunit F17a-A mutant does not bind either. This suggests that the major subunit is needed for the export, the final conformation, or the presentation of F17a-G on the bacterial surface (155).

The Regulation of Fimbrial Expression

Each fimbrial gene cluster typically includes one or two genes that specifically regulate the transcription of the genes in their cluster. In general, it is assumed that fimbrial gene clusters have only one promoter per direction of transcription. When studied in more detail, however, some gene clusters were found to carry multiple promoters and operons for the different accessory proteins (151). In addition to the fimbria-specific regulators, each fimbrial gene cluster typically belongs to specific regulons that can be activated or repressed by global regulators such as H-NS (histone-like nucleoid-structuring protein for temperature and osmolarity-mediated signals) or CRP (cAMP receptor protein for catabolite repression). Fimbrial expression can also be regulated posttranscriptionally. Moreover, some fimbriae undergo phase variation. Transcriptional regulation ensures that all bacterial siblings synchronize their "on" or "off" switch for fimbrial expression. In contrast, a bacterial population regulated by phase variation always contains both "on"- and "off"-switched variants. The ratio of "on" and "off" variants depends on environmental growth conditions, and one type of variant may be as scarce as mutants. It is thought that phase variation improves the survival rate of a bacterial population that is abruptly submitted to a new environment that selects for the scarcer phase variant.

The K88 fimbrial gene cluster carries two genes, faeA and faeB, encoding proteins that are similar to regulatory proteins of the P and S fimbriae (112). FaeA negatively controls K88 expression, whereas FaeB does not seem to regulate K88. cis-active regulation of fimbrial expression is mediated by the level of methylation of three GATC sites upstream of faeB (113, 289). Dam (deoxyadenosine methylase) methylation of the first GATC site prevents the coordinate binding of the global regulator LRP and FaeA at this site, a regulatory mechanism thought to inhibit a lethal overproduction of fimbriae. Methylation of the two other GATC sites destabilizes Lrp/FaeA binding, and methylation of the third site activates transcription of faeB (and downstream fae genes). The population of K88-encoded plasmids in a culture consists of a mixture of replicons that are either methylated (20%, responsible for high-level expression) or nonmethylated (80%, responsible for low-level expression) at the third GATC site (the first site remaining methylated and the second, nonmethylated). Thus, the methylation status of the third GATC site modulates the level of fimbrial expression. The presence of the two IS1 elements between faeA and faeB results in the lack of any detectable regulatory effects of FaeB on faeA or faeB transcription and in the constitutive expression and autoactivation of faeA. These effects were proposed to explain why the K88 fimbriae do not undergo phase variation as observed with the P fimbriae, despite the presence of similar regulatory proteins and GATC sites in cis (113). No regulatory genes have been identified for the F41 fimbriae, but F41 expression is repressed at low temperature and in the presence of alanine, indicating that the K88 and similar F41 fimbrial systems are regulated in a different manner.

The production of K99 fimbriae or the transcription of the major subunit gene fanC is activated in the logarithmic growth phase by oxygen, by low pH, and by glycerol, whereas fewer fimbriae or fan transcripts are expressed in stationary phase, at high pH, and in glucose-, pyruvate-, arabinose-, or lactose-containing media (122, 297). Studies on several compounds as carbon source for bacterial growth indicated that acetate has a strong inhibitory effect on fimbriation and that, together with glucose, it essentially suppresses K99 expression (86). Fimbrial subunit production is drastically reduced in a cya mutant, confirming that K99 is regulated by catabolite repression (125). Curiously, unlike many other fimbrial systems, H-NS is not involved in the noticeable downregulation of K99 expression at low temperature (290, 297). Moreover, two groups of K99-producing ETEC strains were distinguished based on the regulation of fimbriation by different growth conditions (95). Upstream of the K99 gene cluster are two genes, fanA and fanB, that are transcribed in the same direction as the K99 genes and activate K99 expression (228). Studies on K99 gene transcription suggested that FanA and FanB act together as transcriptional antiterminators on two factor-dependent terminator sites after the fanA and fanB sequences (227). A third terminator site after the fanC sequence includes a dyad symmetry for a potential stem-loop structure, suggesting that transcriptional termination at this site is factor independent. Lrp (leucine-responsive regulatory protein) acts as a K99 transcriptional activator by binding to the fanA promoter (31). It is likely that the inhibitory effects of alanine and leucine on fan gene transcription and K99 fimbrial expression (31, 55) are mediated through Lrp. The K99 gene cluster seems to consist of three operons, one for the fanA to fanD genes, a second one for the fanE and fanF genes, and the third one for the fanG and fanH genes (121, 151). A stem-loop structure between fanE and fanF might act as an attenuator of fanF transcription. Putative promoters were mapped for the last two operons and the one upstream of fanE was adjacent to a CRP-binding consensus site, suggesting that the fanEF operon, like the fanABCD operon, is also regulated by catabolite repression.

Early studies recognized that 987P fimbriae are best expressed in vivo in piglet intestines or in vitro when bacteria are grown to stationary phase, forming pellicles at the air-medium interface (128, 198). 987P fimbrial expression undergoes phase variation. Specific environmental signals or growth conditions regulate the rate of phase variation (291). The mechanisms and the potential cis- or trans-active elements regulating 987P phase variation are different from those of the 987P-similar CS18 fimbriae of human ETEC (73, 74, 76, 109). Unlike CS18, no 987P DNA segment is directly regulated by DNA inversion (2, 109). Moreover, dam methylation is not involved in 987P expression or phase variation (76, 288). The apparent stability of the duplicated 987P fimbrial genes in the same clinical strain on a plasmid and on the chromosome (41, 75, 240) may suggest that the merodiploid fas genes confer some advantage to the host strain. Whether the duplicated 987P DNA is required for phase variation in not known. Phase variation being recA-independent, however, any mechanism of intrabacterial DNA exchange explaining phase variation would have to involve other recombinases (75). Expression of the major subunit FasA and of 987P is upregulated by FasH (FapR) (144, 240). FasH shares sequence similarity with the DNA-binding domain of the AraC transcriptional activator and more specifically with the Rns subfamily of positive regulators of fimbriae and other virulence factors of Enterobacteriaceae (193). A portion of the proximal IS1 sequence of the Tn1681 transposon located upstream of fasH is involved in activating fimbrial expression (145). The expression of fasH and fasA are both regulated in response to the carbon source and the nitrogen source (73, 76). Since these nutritional signals are differentially modulated in the intestinal environment, they may provide a mechanism to allow preferential colonization of different segments of the intestine by various enteropathogens (73, 74).

No regulatory genes for F18 or F17 fimbriation have been described yet. F41 expression is temperature regulated (290). Although special growth media seem to improve F18 fimbrial expression in vitro (226), not all the strains express F18 under these conditions (201). In contrast to F18ac, most F18ab fimbriae of clinical isolates are poorly expressed on commonly used media, suggesting a different mechanism of regulation for these two types of fimbriae. The F18 gene cluster is similar to the AF/R1 fimbrial gene cluster of rabbit attaching and effacing E. coli (39). Whether F18 expression is regulated by proteins that are similar to the cis-active transcriptional regulators of AF/R1 remains to be determined.

RESEARCH BENEFITS: DIAGNOSTIC TOOLS, VACCINES, AND INHIBITORS OF COLONIZATION

Studies on ETEC fimbriae have helped to better understand the biology and role of these organelles in pathogenesis; they have also opened the door to new diagnostic, prophylactic, and therapeutic tools. DNA sequence information has led to the development of various molecular approaches, including PCR, for the identification of ETEC fimbrial sequences in clinical isolates (21, 30, 87, 89, 208, 216). Some fimbriae, such as the 987P, are not well expressed when the bacteria are grown in conventional diagnostic media under usual growth conditions. Thus, in comparison with the detection of fimbriae by specific antibodies, PCR is typically a more sensitive technique. However, epidemiological studies on clinical isolates using PCR detection might overestimate the number of strains capable of expressing fimbriae, since the technique might not be able to differentiate intact gene clusters from mutated ones. Conversely, an insufficiently recognized problem of epidemiological studies on pathogenic bacteria whose virulence factors are encoded on mobile DNA elements such as plasmids is that these DNAs can be unstable under in vitro growth conditions (240, 260), resulting in their loss and a concomitant underestimation of ETEC isolate numbers.

Following on the seminal studies of Rutter and Jones (137, 138, 233, 234) demonstrating that colostral antibodies induced by maternal immunization protected neonatal piglets, many additional in vitro and in vivo studies confirmed that fimbriae are highly immunogenic proteins and that the induced antibodies protect by inhibiting adhesion to enterocytes and intestinal colonization (51, 123, 126, 186, 195, 199, 232, 243, 256, 257). Studies with ETEC strains of veterinary relevance have led to the development of effective parenteral antiadhesive vaccines based mainly on three types of fimbriae (180, 202). In addition to its biological effectiveness (181), vaccination of sows was found to be a cost-effective health management strategy (301).

Fimbriae are thought the be very good immunogens because they present to the host a set of epitopes that are repeated 10² to 10³ times on each fimbrial thread, when isolated fimbriae are used as antigen, or 10⁵ to 10⁶ times on each bacterial surface, for fimbriated bacteria. Actually, this property can be used to increase the immunogenicity of foreign epitopes by engineering them into fimbrial subunits and displaying them in a polymeric form on attenuated live bacterial vaccines (15, 44, 45, 46, 225, 274). Thus, it remains to be seen whether vaccine technologies that focus on fimbrial subunit immunogens such as DNA vaccines (278) or edible vaccines (111, 230) will result in protection as effective as that elicited by conformationally mature fimbrial immunogens in large animals. For fimbrial systems whose adhesive and major subunits are different, the question whether a pure adhesin vaccine is more effective than an intact fimbrial vaccine has not been studied in farm animals. Other advantages of assembled ETEC fimbriae are their relative resistance to enteric proteases and their ability to induce mucosal immunity. Formalin-treated fimbriated E. coli, live nontoxigenic E. coli, or attenuated Salmonella enterica serotype Typhimurium expressing cloned fimbriae have been used to immunize animals by the oral route and to successfully induce antiadhesive antibodies (7, 10, 100, 185, 187). Oral vaccinations, combined with parenteral applications, can increase and prolong the duration of lacteal immunity (185). A potential advantage of fimbriae of enteric pathogens is their enteroadhesive properties, which they share with other mucosal immunogens such as the enterotoxins. The binding of fimbriae to complementary intestinal receptors in the appropriate host species is important for the activation of mucosal immunity after oral immunization, as shown with the K88 fimbriae (26, 284). Oral immunization of weaned pigs with these fimbriae was better at priming a mucosal response than intramuscular administration. Induction of a primary immune response occurred only in pigs expressing the corresponding intestinal K88 receptor, suggesting that receptor binding may facilitate antigen uptake (285). The latter pigs were protected against a challenge with K88-fimbriated ETEC. Parenteral priming with K88 induces suppression of a mucosal K88 recall response upon oral infection with K88-fimbriated bacteria (23). In contrast, orally administered K88 was able to prime an immune response in both K88-susceptible and -resistant pigs, indicating that K88 given by the mucosal route does not induce oral tolerance (283).

Passive protection mediated by colostral antifimbrial antibodies fades at weaning and the feeding of anti-F18 antibodies to weaned pigs is protective but expensive and work intensive (305). To protect piglets against postweaning ETEC infections, various active immunization studies have focused on the relevant F18 and K88 fimbriae. Administering nonattenuated live ETEC strains expressing F18ab or F18ac fimbriae to pigs shortly before or after weaning had a protective effect on a second challenge, but most of the vaccinated pigs suffered mild to severe diarrhea from the first immunization (22, 236). Slower colonization of F18-fimbriated ETEC versus K88-fimbriated ETEC paralleled a slower induction of the humoral immune response (293). The oral administration of enteric-coated K88 or microencapsulated F18 fimbriae to newborn piglets at best marginally reduced intestinal colonization upon challenge after weaning (85, 258). All things considered, an efficient vaccine protecting against postweaning ETEC infections awaits further developments, including the design and evaluation of attenuated live bacteria or (and) fimbrial protein vaccines that include adjuvants (4, 287).

Identification and characterization of the binding moieties of ETEC fimbrial adhesins should be useful for the design of new prophylactic or therapeutic strategies. Some studies describing potential receptor or adhesin analogues that interfere with fimbria-mediated colonization have been described (162, 192, 204, 206, 207). However, more studies including efficient inhibition of the relevant panoply of ETEC fimbriae are needed for this approach to be applied in agriculture. Although fat globule membranes of sow and cow milk were reported to contain receptors for ETEC fimbriae (8, 9, 152, 161, 162), the postulated protective role of these receptors in the intestines of young animals remains unknown. Oral administration of proteases that degrade intestinal receptors have been investigated with some success (43, 194). Certain probiotics such as lactobacilli can bind to enterocytes without interfering with the attachment of K88-fimbriated ETEC (261). It was suggested that the coaggregation of certain Lactobacillus isolates with the K88 ETEC decreases ETEC colonization. Probiotics were the most efficient in controlling diarrhea in calves when used in conjunction with fimbria-based vaccines (11). The in vivo relevance of K88-mediated adhesion inhibitors found in certain Lactobacillus culture supernatants remains to be determined (29, 220).

Acknowledgments

I thank Len Bello and Marjan van der Woude for their critical review of this work.

This work was supported by U.S. Department of Agriculture (USDA) Grants 2002-35204-11650 and 2002-35204-12216.

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